An updated phylogeny and adaptive evolution within Amaranthaceae s.l. inferred from multiple phylogenomic datasets

Abstract Amaranthaceae s.l. is a widely distributed family consisting of over 170 genera and 2000 species. Previous molecular phylogenetic studies have shown that Amaranthaceae s.s. and traditional Chenopodiaceae form a monophyletic group (Amaranthaceae s.l.), however, the relationships within this evolutionary branch have yet to be fully resolved. In this study, we assembled the complete plastomes and full‐length ITS of 21 Amaranthaceae s.l. individuals and compared them with 38 species of Amaranthaceae s.l. Through plastome structure and sequence alignment analysis, we identified a reverse complementary region approximately 5200 bp long in the genera Atriplex and Chenopodium. Adaptive evolution analysis revealed significant positive selection in eight genes, which likely played a driving role in the evolution of Amaranthaceae s.l., as demonstrated by partitioned evolutionary analysis. Furthermore, we found that about two‐thirds of the examined species lack the ycf15 gene, potentially associated with natural selection pressures from their adapted habitats. The phylogenetic tree indicated that some genera (Chenopodium, Halogeton, and Subtr. Salsolinae) are paraphyletic lineages. Our results strongly support the clustering of Amaranthaceae s.l. with monophyletic traditional Chenopodiaceae (Clades I and II) and Amaranthaceae s.s. After a comprehensive analysis, we determined that cytonuclear conflict, gene selection by adapted habitats, and incomplete lineage sorting (ILS) events were the primary reasons for the inconsistent phylogeny of Amaranthaceae s.l. During the last glacial period, certain species within Amaranthaceae s.l. underwent adaptations to different environments and began to differentiate rapidly. Since then, these species may have experienced morphological and genetic changes distinct from those of other genera due to intense selection pressure.


| INTRODUC TI ON
Amaranthaceae sensu lato, belonging to the order Caryophyllales within the phylum Angiosperm, is considered a moderately large family that includes both the Chenopodiaceae (this name has been abolished and we now refer to it as traditional Chenopodiaceae) and the Amaranthaceae sensu stricto (Mabberley, 2017).Many economically important plants, such as quinoa (Chenopodium quinoa Willd.), spinach (Spinacia oleracea L.), and sugar beet (Beta vulgaris L.), are predominantly classified under traditional Chenopodiaceae (Alvarez-Jubete et al., 2010;Kupper, 2005;Zhang et al., 2016).These plants provide non-conventional protein sources due to their excellent nutritional value, significantly contributing to human nutrition (Caruso et al., 2018;Pellegrini et al., 2018).Most traditional Chenopodiaceae plants benefit from saline growth media (Akhani et al., 2007;Chen et al., 2020;Shegebayev et al., 2023;Sultanova et al., 2021).A considerable proportion of arable land worldwide is affected by salinity, increasing the demand for salt-tolerant crops (Slama et al., 2015).Most Amaranthaceae s.l.thrive in arid and saline environments, with leaves and bracts adapted into fleshy forms, enabling them to grow in saline habitats and potentially produce useful crops on salty soils (Akhani et al., 2007;Piirainen et al., 2017).The high salt tolerance and bioactive compound content in traditional Chenopodiaceae plants such as Kali Mill.and Salicornia L., along with their functional and health properties, make these glassworts important candidates for future fresh and processed foods (Karakas et al., 2017;Ventura et al., 2015).
Although Amaranthaceae s.l.provides abundant nutrition and protein as crops, the phylogenetic relationships between Amaranthaceae s.s. and traditional Chenopodiaceae remain unsatisfactorily resolved due to morphological similarities at the species-level and ILS events at the molecular level (Bao et al., 2003;Frankton, 1968;Huang et al., 2020;Tutin, 1964).Correct classification of Amaranthaceae s.l.taxa is essential to utilize traditional Chenopodiaceae as raw materials for economically important plants and crops.Recent molecular systematic studies have confirmed the monophyly of both Amaranthaceae s.s. and Chenopodiaceae, indicating their close relationship (The Angiosperm Phylogeny Group, 2016; Yang et al., 2018;Yao et al., 2019).However, the phylogenetic position of Betoideae remains unresolved, leading to ongoing debates about Chenopodiaceae.Consequently, scholars have considered Chenopodiaceae an untenable family, resulting in its merger with Amaranthaceae s.s.(Brignone et al., 2021;Mabberley, 2017;The Angiosperm Phylogeny Group, 2016).Amaranthaceae s.l.faces issues in species classification and phylogenetic relationships between lineages in current systematic studies.
We supplemented molecular data of Amaranthaceae s.l.worldwide by collecting local dominant plant groups.We hypothesized that different climate conditions and habitat characteristics specific to each region have influenced the evolution of genes in Amaranthaceae s.l., leading to adaptive differentiation.Against this background, we studied the systematic evolution of Amaranthaceae s.l.using plastomes and nuclear ribosomal DNA collected from the Qaidam Basin and previously published data.Therefore, we aimed to clarify (1) the phylogenetic relationship between the two major lineages of Amaranthaceae s.l.(Amaranthaceae s.s. and traditional Chenopodiaceae); (2) to identify protein-coding genes (PCGs) that contributed to adaptive divergence and explore the significance of these genes on the evolution of Amaranthaceae s.l.; and (3) to infer the diversification of Amaranthaceae s.l.through molecular and temporal evolution.

| Plant sources
We collected young and fresh leaves from 21 Amaranthaceae s.l.samples, comprising 20 samples from the saline-alkali environments of the Qaidam Basin in Qinghai Province and one sample from Gaize County in the Tibetan Autonomous Region, each representing one individual species (Table 1).The plant materials were dried in silica gel and stored at −20°C.Vouchers for all the samples were deposited into the Qinghai-Tibetan Plateau Museum of Biology (QTPMB), University of Chinese Academy of Sciences.Additionally, we included plastomes of non-replicated species from earlier studies in this analysis (Table S1).

| DNA extraction and sequencing
Total genomic DNA was extracted from Amaranthaceae s.l.leaves using the modified CTAB protocol of Yang et al. (2014).We determined DNA concentration using a microspectrophotometer (Nanodrop 2000 and Qubit 4, USA) and DNA quality using 1% agarose gel electrophoresis.
After the library was finished and the quality controlled, sequencing was performed on an Illumina NovaSeq 6000 platform (Illumina Inc., San Diego, CA, USA) at Novogene in Tianjin, China, generating 10 Gb of high-quality 150-bp paired-end reads per sample.

| Sequence assembly and annotation
We filtered the raw sequence reads using Trimmomatic v.0.38 (Bolger et al., 2014) to remove adaptors and low-quality sequences with the | 3 of 17 XU et al.

| Genome structure and comparative analysis
Plastome characteristics, including the number of genes, nucleotide polymorphism, and structural diagrams, were analyzed.We visualized the divergence of 59 complete plastomes using the mVISTA online genome comparison tool (Frazer et al., 2004).Nucleotide diversity (Pi) was calculated using 79 de-redundant PCGs and 127 intergenic spacer regions with DnaSP v.6.12 (Rozas et al., 2017).

| Adaptive evolution detection
To investigate positive selection in Amaranthaceae s.l., we analyzed the ratio (ω = d N /d S ) of non-synonymous (d N ) and synonymous (d S ) mutations using PAML v.4.9j (Yang, 2007).Maximum likelihood (ML) phylogenetic trees were reconstructed with IQ-TREE v.2.0.3 (Nguyen et al., 2015) using a best-fit model obtained by ModelFinder v.1.6.12 (Kalyaanamoorthy et al., 2017) with 10,000 bootstrap replicates.Site models (seqtype = 1, model = 0, NSsites = 1, 2, 7, 8) in CodeML from PAML v4.9j were used to identify positively selected sites in protein-coding regions.Likelihood ratio tests (LRT) using the site models (including M1 [neutral] vs. M2 [positive selection] and M7 [beta] vs. M8 [beta and ω]) and the branch-site models (including null hypothesis; Yang & Nielsen, 2002) identified positive sites with significant posterior probability support (p ≥ .99)and compared values of different models (Whelan & Goldman, 1999).Branch-site models were employed to further assess genes identified as under selection in site models, testing the alternative hypothesis using the branch of traditional Chenopodiaceae (Clades I and II) as the foreground branch, with the corresponding null hypothesis (ω = 1) TA B L E 1 Sample collection information and GenBank accession for plastomes of Amaranthaceae s.l.examined for likelihood ratio.Positive selection sites were identified using the BEB method (Yang et al., 2005).
Both BI and ML analyses were performed identically for all datasets.
For the coalescence-based method, we constructed 81 singlegene ML trees based on dataset (i) shared across all species using IQ-TREE v.2.0.3 (st = CODON11, m = MFP) and merged these tree files to reconstruct a species tree using ASTRAL v.5.7.8 (Zhang et al., 2018).Using the 81 shared PCGs, we examined discordance between gene trees and the species tree with Phyparts v.0.0.1 (Smith et al., 2015).We used Toytree v.2.0.1 (Eaton, 2020) to build cloud tree plots to display conflicts between the single-gene trees and the species tree.

| Molecular features of the plastomes
We analyzed the complete plastomes of 59 Amaranthaceae s.l.
taxa and five outgroups, revealing a typical circular tetramerous structure comprising a large single copy (LSC), a small single copy (SSC), and two inverted repeats (IRs; Table S1).The total length of Amaranthaceae s.l.plastomes was similar, ranging from 149,722 to 155,108 bp, and the GC content remained stable, varying from 36.2 to 37.6%.Most species had a highly identical genetic composition in terms of gene order and number.The number of PCGs encoded by plastomes ranged from 84 to 87, with subtle variation due to the presence or absence of ycf15 (Table 2).The number of transfer RNA genes was mostly 37, although a few species had lost trnS GCU , trnG-GCC , trnN GUU , and others due to genetic mutations.Ribosomal RNA genes were highly conserved and had not changed in number.

| Structural and sequence divergence analysis of the plastomes
We used the plastomes of 13 species of Amaranthaceae s.l. as representatives of their tribes to compare the characteristics of plastomes among these species (Figure 1).Our finding showed that two IR regions were more conserved than the other two regions, and coding regions displayed low divergence in sequence characteristics.We also found that two tribes (Atripliceae and Chenopodieae) had about 5200 bp of the reverse complementary region in the middle and downstream of the LSC regions, involving the tandem gene sequence (rbcL-atpB-atpE-trnM CAU -trnV UAC ; Figure S1).Nucleotide diversity (Pi value) was detected separately for PCGs and non-coding regions (Figure S2).Pi values of PCGs ranged from 0.0074 (ndhB) to 0.3934 (ycf15), indicating large sequence diversity among different tribes in Amaranthaceae s.l.
individuals for positive selection, excluding duplicated genes in IRs.
This identified eight genes that were positively selected through statistical analysis (Table 3).These genes included hypothetical protein reading frame genes 1 and 2 (ycf1 and ycf2), accD, ccsA, ndhA, rbcL, rpl22, and rps12.Based on M2 (positive selection) and M8 (beta and ω) site models, we identified positive selection sites within these genes: ycf2 contained 16 and 18 sites, ycf1 contained 15 and 15 sites, accD contained 5 and 5 sites, rpl22 contained 3 and 3 sites, rbcL contained 3 and 7 sites, ccsA contained 1 and 2 sites, ndhA contained 1 and 3 sites, and rps12 contained 1 and 1 sites (Table S3).The branchsite models with traditional Chenopodiaceae as foreground branches identified that ycf2 and ycf1 each contained 16 sites, accD, rpl22, and rbcL each contained 4 sites, ndhA contained 2 sites, and rps12 contained 1 site, while ccsA did not contain any sites (Table S4).Both the branch-site models and the site model exhibited highly similar results, with statistically significant positively selected sites (p < .01).

| Phylogenetic relationships of Amaranthaceae s.l.
To effectively assess the evolutionary information in nucleotides, we used five datasets to determine the phylogenetic relationships within Amaranthaceae s.l.using ML and BI statistics.

Celosia argentea ycf15
Celosia cristata ycf15 (Continues)  We also compared the coalescent and gene phylogenies, indicating discordance between some gene trees and the species tree within the plastome, as indicated by the Clades of Atriplex, Amaranthus, Salsoleae, and traditional Chenopodiaceae (Figure 4a).

| Divergence time estimation
The molecular divergence of Amaranthaceae s.l. and its sister taxa  S5).The crown age of Amaranthaceae s.l.Most research has shown that differences in plastome sizes between species are mainly reflected in the boundaries of the IR region (Olmstead & Palmer, 1994).The expansion and contraction of the IR region significantly affect plastome size (Chumley et al., 2006;Zhang et al., 2014).The variation in the IR region length of traditional Chenopodiaceae species tends to be small, indicating that the boundary of the IR region is relatively conserved in the adaptive evolution of Amaranthaceae s.l.(Hong et al., 2017).For genes in plastomes, the number and type of rRNA are stable, but there are a few differences in tRNA.Nucleotide mutations in individual species led to the termination of transcription, such as trnG GCC , trnN GUU , trnS GCU , and others (Table 2).However, these differences have little impact on the evolution of species, as they contain minimal evolutionary information.In contrast, variations in the PCG regions can cause changes in the evolutionary information of species.For instance, more than half of Amaranthaceae s.l.lack ycf15 in the plastomes (Table 2).
Comparing the reference sequence of ycf15 with plastomes lacking this sequence, we found that gene loss could be due to deletions or substitutions of loci in certain fragments (Figure S7).Ycf15 belongs to a family of PCGs that originated from eukaryotes or horizontal transfer (Martin et al., 1998;Stegemann et al., 2012).Our results suggested that this may contribute to differences among plants and may be one reason for the inconsistency between molecular data and morphology.
The arrangement of internal segments in plastomes plays a significant role in multiple evolutionary events during the radiation of Amaranthaceae s.l.In Mammillaria Haw.plants (Cactaceae) of the Caryophyllales, there was a block of genes rearranged in the LSC region of the plastomes.Although not all Mammillaria species had the same rearrangement order, some share the same order with Carnegiea Perkins and Pachycereus Britton & Rose (both genera belonging to Cactaceae; Solórzano et al., 2019).Through comparative genomic analysis, it was found that there was a 5200 bp reverse complementary fragment in the genera Chenopodium L. and Atriplex compared to other Amaranthaceae s.l.This is similar to the situation we found in the LSC region of Atriplex and Chenopodium, where the plants of each genus were not completely distinguished in phylogenetic results.This rearrangement block may be a synapomorphy in tribes Atripliceae and Chenopodieae during the radiation evolution of the subfamily Chenopodioideae, suggesting that the arrangement of internal segments of plastomes cannot be ignored.
The plastome is maternally inherited in most angiosperms and is widely used due to its advantages in simple sequencing and assembly, conserved sequences, stable structure, and no interference The topology of the species tree based on coalescence shows discordance among 81 shared PCGs (cloud tree).(a) The heavy black line represented the species tree constructed by Astral, and the thin lines represented gene trees constructed by shared PCGs.(b-d) Topology of the three major branches (Clades I, II, and Amaranthaceae s.s.) in 81 shared genes of Amaranthaceae s.l.Thick-solid lines indicated branches containing all or most species, while thin-dotted lines indicated species clustering outside of the branch with few or no members.Three gene trees were not included due to their excessively complicated topology.Pie charts on the nodes showed the degree of concordance between species and gene trees, where the red and blue parts represent discordance and concordance, respectively.from genetic recombination (Jansen et al., 2007;Luo et al., 2016).
Therefore, hotspots of variation in plastomes are easy to observe, and regions with higher hotspots are often used as molecular markers to help correctly distinguish taxon and effectively protect wild plant resources.To analyze the nucleotide diversity of 79 deredundant PCGs and 127 intergenic spacer regions of 59 species of Amaranthaceae s.l., we employed DnaSP and mVISTA to compare the complete plastomes.The results showed that changes in the intergenic spacer regions are significantly greater than those in the coding regions (Figures 1 and S2).This is consistent with previous studies using short orthologous DNA sequences (DNA barcodes) containing evolutionary information to analyze the phylogeny of Amaranthaceae s.l.(Kress et al., 2005).However, these studies have some problems, such as low support rates and difficulties in | 11 of 17 effectively distinguishing species.The reason for these issues may be that the nucleotide diversity of loci such as rbcL (0.0414), atpB-rbcL (0.0738), and psbB-psbH (0.0766) used in prior research is lower than the average nucleotide diversity of PCGs (0.05) and intergenic spacer regions (0.0829), respectively (Kadereit et al., 2003(Kadereit et al., , 2010;;Kadereit & Freitag, 2011;Schüssler et al., 2017).Therefore, the diversity hotspots obtained in this study can provide insights on how to better classify species with controversial classification within Amaranthaceae s.l.

| Adaptive evolution
During the evolution of Amaranthaceae s.l., we identified eight positively selected genes.Ycf1 and ycf2 encode components of the plastome inner envelope membrane protein, crucial for cell survival.They are also the two largest open reading frames in plastomes (Drescher et al., 2000;Kikuchi et al., 2013).In the plastomes of Amaranthaceae s.l.examined in this study, ycf1 gene spans approximately 5400 bp, extending across the SSC and IR regions.Due to a small segment of ycf1 (approximately 1000 bp) being located on the IR side, its reverse complementary property results in a 1000 bp ycf1 pseudogene within another IR region.This pseudogene is only 1/5 the length of ycf1 due to premature stop codons, ultimately resulting in its lack of functional characteristics of ycf1.Prior research has reported strong positive selection effects of these two genes in Quercus and Caragana (Jiang et al., 2018;Yang et al., 2016).Among other positive selected loci, accD, a gene encoding the β-carboxyl transferase subunit of acetyl-CoA carboxylase, maintains the plastome compartment and has ACCase that synthesizes substances necessary for leaf development (Kode et al., 2005).Additionally, ccsA encodes a protein required for heme attachment to c-cytochromes (Xie & Merchant, 1996).Thus, these four genes improve the oxidation of Amaranthaceae s.l.plant cells in extreme environments by betterutilizing oxygen.The remaining positively selected genes have more or less beneficial effects on plant cells.Environment selection enables species to adapt to complex and variable habitats.Previous studies have also reported positive selection of these genes in plants such as Gentiana L., Bignoniaceae, and Amaranthaceae, which have adapted to adversity, evolving characteristics suitable for surviving in harsh environments (Kapralov et al., 2012;Sobreiro et al., 2020;Zhou et al., 2018).
Intriguingly, the branch-site models, with traditional Chenopodiaceae as the foreground branch, did not detect any positively selected amino acid sites in ccsA, and its LRT-test results were not particularly significant (.01 < p < .05,Table S4).The positively selected sites in the remaining seven genes were highly similar to the site model results, with significant differences in LRT-test and ω > 1.
The phylogenetic topology of these seven positively selected genes mainly formed ((Clades I and II), Amaranthaceae s.s., Table S6), suggesting that they may be one of the driving forces for the evolution of traditional Chenopodiaceae into a monophyletic group.The adaptive evolution of these seven genes in traditional Chenopodiaceae Clade has played an important role, providing insight into the adaptive evolution of Amaranthaceae s.l. and further elucidating chloroplast genetic characteristics.
Ycf15 is a gene in plastomes that has drawn much attention from researchers due to its paradoxical function and evolution (Chumley et al., 2006;Raubeson et al., 2007).Shi et al. explored the function and evolution of the ycf15 gene in angiosperms based on plastomes of Camellia L. species and suggested that ycf15 contains certain phylogenetic information sites causing the divergence topology of branch ends (Shi et al., 2013).However, there is no direct molecular evidence currently suggesting that ycf15 is involved in adaptive evolution.In this study, we mapped the presence of ycf15 onto the phylogenetic tree of Amaranthaceae s.l.(Figure 2).Our results indicate that all Atriplex plants lack ycf15, and all Chenopodium plants contain ycf15 except for C. karoi, which is the closest genus to Atriplex.
Similarly, in the Salsoloideae Clade, the plastomes of Halogeton arachnoideus, Haloxylon persicum, Kali zaidamicum, and Climacoptera obtusifolia include ycf15, but its closely related species do not.The species were collected from areas with severe climatic conditions.
The saline-alkali environment, water shortage, and strong sunlight may limit the growth of halophytes, and the genes may evolve in response to the environment, forming characteristics that differ from the related species (Cheng et al., 2021;Guan et al., 2015;Tao et al., 2017).In addition, the absence of ycf15 within the plastomes of Amaranthaceae s.s. is one of the features that distinguish these plants from traditional Chenopodiaceae.This study speculates that ycf15 in Amaranthaceae s.l. may undergo variation in response to extreme environments.

| Phylogeny and molecular dating analysis of Amaranthaceae s.l.
Previous researchers have conducted extensive molecular systematics studies on Amaranthaceae s.l.(Hernández-Ledesma et al., 2015;Kadereit et al., 2003).However, controversies still exist regarding the systematic relationships between the main lineages of the family (Hammer et al., 2015;Masson & Kadereit, 2013).Based on concatenated PCGs, our study shows that traditional Chenopodiaceae is a monophyly (Clades I and II), while Amaranthaceae s.s.forms an independent clade and is a sister taxon to traditional Chenopodiaceae (Figure 2).Coalescence-based simulation plots revealed discrepancies between some phylogenetic topologies of cloud trees and species tree trees (Figure 4a).Different genes within species may evolve diversely, leading to a large number of ILS events, such as ITS trees with low support rates (Figure 3).Although a few gene trees support Clades I and II as sister groups, their support rates are high.
Statistical analysis of the topology of these gene trees (Figure 4b-d, However, the phylogenetic position of these two species remains ambiguous in the analyses based on both coalescent simulation and full-length ITS (Figure 3).Thus, Chenopodium is not a monophyletic group due to the existence of C. karoi, which is strongly supported For subfamily Salsoloideae in Clade II, we found that the genera Halogeton and Subtr.Salsolinae are not monophyletic lineage.

Akhani et al. divided the conflicted taxonomic relationships within
Salsola into multiple genera based on molecular and morphological information (Akhani et al., 2007).Wen et al. proposed that Halogeton was polyphyletic, and Kali collinum and Ka.zaidamica were not in the same clade as Xylosalsola arbuscula (Pall.)based on ITS and plastome fragments (Wen et al., 2010).Our phylogenetic results also verified that Subtr.Salsolinae was not a monophyletic lineage (Figure 4).The Subtr.Salsolinae species may have developed certain morphological characteristics to adapt repeatedly to drought and saline environments through radiation evolution, such as central sclerenchyma and reduction of surface area in leaves, which promote their C4 cycle and improve productivity under arid conditions (Lauterbach et al., 2017;Schüssler et al., 2017).Therefore, it is recommended that Subtr.Salsolinae should be split into multiple subfamilies to conform to correct taxonomic classification.

| Divergence of estimation of Amaranthaceae s.l.
We provide the origin and molecular evolution time of  S5).This estimate is approximately the median value of previous studies, which estimated the origin time of Amaranthaceae s.l.using rbcL and atpB-rbcL internal spacer regions (47-87 Mya; Kadereit et al., 2012;Magallón et al., 2015).Magallón et al. used five plastid and nuclear markers to conclude that the stem age of Amaranthaceae s.l.occurred at 64.2 or 76.4 Mya (Kadereit et al., 2012;Magallón et al., 2015).Moreover, it was interesting to note that positively selected genes played a driving role in the evolution of Amaranthaceae s.l.The divergence time tree based on these genes confirms the monophyly of traditional Chenopodiaceae and explains the presence of numerous ILS events within the plastome that have previously hindered accurate inference of Amaranthaceae s.l.phylogeny.
We also found that Clades I and II differentiated rapidly in the early Paleocene (61.27 Mya), indicating that broad-scale evolutionary patterns were evident within a clade radiation.The extinction event at the end of the Cretaceous led to the death of many organisms due to their inability to adapt to drastic climate change, thus allowing the surviving plants to reproduce rapidly and occupy available niches (Fawcett et al., 2009;Meredith et al., 2011;Schulte et al., 2010).During periods of less than one Mya or two to three Mya, the Qaidam Basin experienced several stages of the penultimate glacial maximum, last glacial maximum, and interglacial periods , selecting GTR as the substitution model with a strict clock model and a birth-death model of speciation, based on jModelTest2 (Posada, 2008) results indicating the best model as GTR + F + I + G4.Three well-dated fossils were used to calibrate relevant nodes.The stem node of Chenopodieae (Clade I) was calibrated using the Lower Miocene seed fossil of Parvangula randeckensis Hiltermann & Schmitz with a lognormal distribution (mean = 1.0,SD = 0.5, and offset = 23.3Ma; All datasets, except for datasets iii (full-length ITS) and v (non-positively selected PCGs), recovered the monophyly of traditional Chenopodiaceae (Clades I and II; Figures 2, S3 and S4).The three instances of clear cytonuclear discordance are as follows: (a) In the PCGs dataset, C. karoi is sister to Atriplex but is separated from Atriplex in the full-length ITS dataset, TA B L E 2 Statistical analysis of missing genes for Amaranthaceae s.l.
Axyris L., Dysphania R.Br., Krascheninnikovia, and Spinacia were well resolved in the PCGs dataset as belonging to Chenopodieae, but they were placed independently from Chenopodieae and Corispermeae in the full-length ITS dataset, and (c) Beteae belonged to Clade I but is the ancestor of Amaranthaceae s.l. in the full-length ITS dataset (Figure 3).The full-length ITS phylogenetic trees showed relatively low bootstrap values (BS)/posterior probability (PP) in these three discordances.In the Salsoloideae subfamily, Haloxylon Bunge is nested in the genera Halogeton C. A. Mey., and genera Halogeton and Kali did not form monophyletic groups.F I G U R E 1 mVISTA-based visualization of whole plastomes comparison for Amaranthaceae s.l. 13 species was selected to represent their tribes respectively to compare the characteristics of plastome (Be = Beteae, Ch = Chenopodieae, At = Atripliceae, Go = Gomphreneae, Sa = Salsoleae, Ac = Achyrantheae, Ce = Celosieae, Su = Suaedeae, Ae = Aeryeae, Am = Amarantheae, Co = Corispermeae, Ha = Halopeplideae) and Beta vulgaris subsp.vulgaris was used as the reference.TA B L E 3 Likelihood ratio tests (LRT) for eight positively selected sites based on the site models and branch-site models (M1: Neutral; M2: Selection; M7: Beta; M8: Beta & gamma; Model A: The branch-site models; Model null: Null hypothesis).

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was estimated to be 62.55 Mya), coinciding with the end of the Cretaceous and the beginning of the Paleocene.During this stage, the former began to differentiate into Clades I, II, and Amaranthaceae s.s.around the Cretaceous-Paleogene (K-Pg) boundary, whereas traditional Chenopodiaceae experienced a rapid differentiation event, mainly reflected in the short differentiation time branches of traditional Chenopodiaceae.The analysis of adaptive evolution indicated that the divergence time of non-positively selected PCGs was delayed by approximately 20 million years compared to the PCG results.The topology supported the sister relationship of Amaranthaceae s.s. to Clade I (Figures S5 and S6).Meanwhile, the results of positively selected genes were consistent with the PCG results and supported the monophyly of traditional Chenopodiaceae.Gene content and structure of Amaranthaceae s.l.plastomes are conserved generally Accurate identification of plants is vital for the development and breeding of Amaranthaceae s.l., not only for protecting genetic resources but also for sustaining the use of diversity(Flowers et al., 2010;Hooper et al., 2005;Myers et al., 2000).In this study, we F I G U R E 2 Phylogenetic tree of the family Amaranthaceae s.l.revealed by the concatenated datasets of 87 protein-coding genes using Maximum Likelihood (ML) and Bayesian inference (BI).Support values above the branches are ML bootstrap values/Bayesian posterior probabilities.Nodes without digits indicate 100% bootstrap value/1.0posterior probability.reported 21 complete plastomes of Amaranthaceae s.l., ranging in size from 150,585 to 153,832 bp, which falls within the typical range of angiosperm plastomes (120-180 kb)(Goulding et al., 1996;Zhang et al., 2012).Our published plastome sizes align with previously published plastomes of Amaranthaceae s.l.(149,722-155,108 bp;Yao et al., 2019).

F I G U R E 3
Comparison of the Amaranthaceae s.l.plastid and full-length ITS phylogenies.The topologies of (a) and (b) were reconstructed using maximum likelihood (ML) based on 87 PCGs and full-length ITS sequences, respectively.The branches are labeled with the values of BS/PP probability, those above 90/0.90are not displayed.Dotted lines connect the same species between (a) and (b).
Fuentes, Uotila & Borsch and Oxybasis Kar.& Kir. were still controversial, leading researchers to suggest the establishment of additional lineages or merging Atripliceae and Chenopodieae (Fuentes-Bazan, Mansion, & Borsch, 2012).Another study based on trnL-F and matK suggested that the four branches of Chenopodioideae and the core group of Chenopodium should form a monophyletic group called the tribe Atripliceae s.l.(this genus name has priority over Chenopodieae; Fuentes-Bazan, Uotila, & Borsch, 2012).Morphologically, C. karoi differs from the genus Chenopodium in having leaves abaxially with white powder and seed surface with honeycomb pits.We cannot Amaranthaceae s.l.based on plastomes, positively selected genes, and non-positively selected genes, showing that the stem divergence time of Amaranthaceae s.l.occurred during the Paleogene period around 69.80 Mya near the K-Pg boundary (Figures 5, S5 and S6, Table

Table S6
that traditional Chenopodiaceae constitutes a monophyletic group, which contradicts the notion that traditional Chenopodiaceae and Amaranthaceae s.s.should be included in Amaranthaceae s.l.based on APG IV.
) shows that several Amaranthaceae s.s.species clustered within traditional Chenopodiaceae or a small number of traditional Chenopodiaceae species clustered within Amaranthaceae s.s., but this did not affect the primary topology ((Clades I and II), Amaranthaceae s.s.), which is supported by most gene trees.This provides compelling molecular evidence Additionally, our results are consistent with the above-mentioned studies in terms of intergroup relationships, and plastid PCGs are more conservative than transcriptomes, which can enhance the results of Yang and Walker's studies.For Clade I, the primary taxonomic contradiction lies between the genera Chenopodium and Atriplex based on concatenated phylogeny.Our results indicate that, compared to Chenopodium, the relationships between C. karoi and Atriplex are closer, and C. hybridum (L.) is the ancestral species of the two genera (Atriplex and Chenopodium).